The toxin producing bacterium Clostridium difficile causes an estimated 1,000,000 to 3,000,000 cases of diarrhea and colitis in the United States each year at an annual cost of $1.1 Billion dollars for nosocomial infections alone. Most cases are associated with the administration of broad-spectrum antibiotics. It has been assumed that the administration of antibiotics causes changes in the normal gastrointestinal microbiota. These changes allow overgrowth of C. difficile, which has either been present in low numbers in the gastrointestinal tract before the administration of antibiotics, or is acquired from the environment during antibiotic administration. Furthermore, it is likely that C. difficile spores and not the vegetative bacilli are the direct contagion analogous to other pathogenic Clostridia sp. Spores are hardy, not easily cleared from the body by natural means and are resistant to antibiotics. We theorize that the normal gastrointestinal microbiota interferes with gut colonization by C. difficile and may also regulate expression of toxin by the organism. Additionally, we hypothesize that the spore morphotype itself contributes heavily to not only the initial introduction of C. difficile to the host, but also to recurrent relapse and shedding/transmission. To address these hypotheses, three specific aims are proposed for this project. In the first aim, we will compare the fecal microbiota in asymptomatically colonized individuals and patients with initial or recurrent C. difficile infection of varied severity. We will determine if specific community structures correlate with susceptibility to disease and with clinical outcomes/severity. The second aim will extend these observations in a murine model of C. difficile infection to define the microbiologic factors that contribute to colonization resistance against C. difficile. In the third aim the contribution of germination and sporulation to clinical disease and transmission will be determined. We will characterize clinical C. difficile isolates for sporulation/germination attributes and test naturally occurring variants and defined mutants in the murine C. difficile model. These aims will provide important insight in the roles of microbial ecology and molecular bacterial pathogenesis in C. difficile infection.